Involvement of inducible nitric oxide synthase in inflammation-induced dopaminergic neurodegeneration.

The loss of dopaminergic neurones in the substantia nigra with Parkinson's disease may result from inflammation-induced proliferation of microglia and reactive macrophages expressing inducible nitric oxide synthase (iNOS). We have investigated the effects of the supranigral administration of lipopolysaccharide on iNOS-immunoreactivity, 3-nitrotyrosine formation and tyrosine hydroxylase-immunoreactive neuronal number, and retrogradely labelled fluorogold-positive neurones in the ventral mesencephalon in male Wistar rats. Following supranigral lipopolysaccharide injection, 16-18 h previously, there was intense expression of NADPH-diaphorase and iNOS-immunoreactivity in non-neuronal, macrophage-like cells.

Hydrogen peroxide-mediated phosphorylation of ERK1/2, Akt/PKB and JNK in cortical neurones: dependence on Ca(2+) and PI3-kinase.

Primary cortical neurones exposed to an oxidative insult in the form of hydrogen peroxide (H(2)O(2)) for 30 min showed a concentration-dependent increase in oxidative stress followed by a delayed NMDA receptor-dependent cell death measured 24 h later. Extracellular signal-regulated protein kinase (ERK1/2), c-jun N-terminal kinase (JNK) and the kinase Akt/PKB may regulate neuronal viability in response to oxidative insults. Using phospho-specific antibodies, a 15-min stimulation of neurones with H(2)O(2) (100 microm - 1 mm) produced a concentration-dependent phosphorylation of ERK1/2 and Akt/PKB that was partly dependent on extracellular Ca(2+) and phosphatidylinositol 3-kinase (PI3-K).

Chronic high dose L-DOPA alone or in combination with the COMT inhibitor entacapone does not increase oxidative damage or impair the function of the nigro-striatal pathway in normal cynomologus monkeys.

Parkinson's disease (PD) is characterised by a loss of pigmented dopaminergic neurones in the zona compacta of substantia nigra. The mechanisms underlying nigral cell death remain unknown but may involve oxidative damage. There has been concern that L-DOPA treatment may accelerate nigral pathology in PD through chemical and enzymatic oxidation to reactive oxygen species.

The kinetics of hypoxanthine transport across the perfused choroid plexus of the sheep.

The uptake of principal salvageable nucleobase hypoxanthine was investigated across the basolateral membrane of the sheep choroid plexus (CP) perfused in situ. The results suggest that hypoxanthine uptake was Na+-independent, which means that transport system on the basolateral membrane can mediate the transport in both directions. Although the unlabelled nucleosides adenosine and inosine markedly reduce the transport it seems that this inhibition was due to nucleoside degradation into nucleobases in the cells, since non-metabolised nucleoside analogue NBTI did not inhibit the transport.

CB(1) receptor antagonist SR141716A increases capsaicin-evoked release of Substance P from the adult mouse spinal cord.

Cannabinoids have an antinociceptive action in many pain models. We have investigated a possible modulatory role for Type 1 Cannabinoid receptors (CB(1)) on the release of excitatory transmitter Substance P from the adult mouse spinal cord after stimulation of nociceptor terminals by capsaicin. Capsaicin (0.

Effect of overexpression of BCL-2 on cellular oxidative damage, nitric oxide production, antioxidant defenses, and the proteasome.

Bcl-2 is a gene family involved in the suppression of apoptosis in response to a wide range of cellular insults. Multiple papers have suggested a link between Bcl-2 and oxidative damage/antioxidant protection. We therefore examined parameters of antioxidant defense and oxidative damage in two different cell lines, NT-2/D1 (NT-2) and SK-N-MC, overexpressing Bcl-2 as compared with vector-only controls.

6-Hydroxydopamine-lesioning of the nigrostriatal pathway in rats alters basal ganglia mRNA for copper, zinc- and manganese-superoxide dismutase, but not glutathione peroxidase.

The effects of nigrostriatal pathway destruction on the mRNA levels of copper, zinc-dependent superoxide dismutase (Cu,Zn-SOD), manganese-dependent superoxide dismutase (Mn-SOD), and glutathione peroxidase in basal ganglia of adult rat were investigated using in situ hybridization histochemistry and oligodeoxynucleotide (single-stranded complementary DNA) probes. The 6-hydroxydopamine (6-OHDA)-induced destruction of the nigrostriatal pathway resulted in contralateral rotation to apomorphine and a marked loss of specific [(3)H]mazindol binding in the striatum (93%; P<0.05) and of tyrosine hydroxylase mRNA in substantia nigra pars compacta (SC) (93%; P<0.

Chronic high dose L-dopa treatment does not alter the levels of dopamine D-1, D-2 or D-3 receptor in the striatum of normal monkeys: an autoradiographic study.

To assess the role of dopamine receptors in the genesis of dyskinesia, we have used quantitative autoradiography to determine the effect of chronic L-dopa administration on dopamine D-1 (using [3H]SCH 23390), D-2 (using [3H]spiperone) and D-3 (using [3H]7-OH-DPAT) receptor binding levels in the striatum of dyskinetic or non-dyskinetic monkeys. Total and subregional striatal analysis showed no difference in D-1, D-2 or D-3 receptor binding in the caudate and putamen between monkeys receiving high dose L-dopa treatment with marked dyskinesia and those without dyskinesia compared to untreated animals. It thus appears unlikely that changes in dopamine receptor expression are a primary cause of L-dopa induced dyskinesia.

Endogenous galanin is required for the full expression of central sensitization following peripheral nerve injury.

The neuropeptide galanin is known to be involved in nociceptive sensory processing in the spinal cord. We have attempted to better characterise the function of endogenous galanin in nociceptive signalling by examining a mouse strain carrying a loss of function mutation in the galanin gene (gal-/-). Galanin expression is significantly up-regulated following damage to a peripheral nerve.

Development of the fallopian canal in humans: a morphologic and radiologic study.

Aims: This study investigated the development of the fallopian canal with particular reference to the mode of ossification and dehiscences, sites of incomplete closure around the facial nerve.

Background: The precise sequence of events surrounding ossification of the tissues around the facial nerve is uncertain. Incomplete ossification results in dehiscence of the adult structure, which places the nerve at increased risk of damage from disease processes in the middle ear and iatrogenic trauma during otologic surgery.

Lack of influence of dietary nitrate/nitrite on plasma nitrotyrosine levels measured using a competitive inhibition of binding ELISA assay.

The action of peroxynitrite in vivo has been proposed to account for the involvement of nitrotyrosine in the pathogenesis of many diseases. However, it has been demonstrated that nitrite under acidic conditions, similar to those in the human stomach, also has the ability to nitrate tyrosine. Dietary nitrate is also implicated in the progression of gastritis and gastric cancer and elevated levels of nitrate are found in many disease states in which nitrotyrosine may play a role.

The mechanisms for nitration and nitrotyrosine formation in vitro and in vivo: impact of diet.

The detection of 3-nitro-L-tyrosine residues associated with many disease states, including gastric cancer, has implicated a role for peroxynitrite in vivo, and thus endogenously produced nitric oxide and superoxide. Additionally, dietary nitrate has been suggested to be involved in the pathogenesis of gastric cancer through a mechanism involving reduction to nitrite and subsequent formation of potentially mutagenic nitroso-compounds. Studies have now demonstrated that a multitude of reactive nitrogen species other than peroxynitrite are capable of producing nitrotyrosine.

A role for the TTX-resistant sodium channel Nav 1.8 in NGF-induced hyperalgesia, but not neuropathic pain.

The tetrodotoxin-resistant voltage-gated sodium channel Nav 1.8 is expressed only in nociceptive sensory neurons. This channel has been proposed to contribute significantly to the sensitization of primary sensory neurons after injury.

Flavonoids protect neurons from oxidized low-density-lipoprotein-induced apoptosis involving c-Jun N-terminal kinase (JNK), c-Jun and caspase-3.

Oxidative stress has been associated with neuronal loss in neurodegenerative diseases and during age-associated cognitive decline. Flavonoids have been proposed to play a useful role in protecting the central nervous system against oxidative and excitotoxic stress, although the mechanism of action is unknown. Using oxidized low-density lipoprotein (oxLDL) as the oxidative insult we investigated the mechanism of neurotoxicity and attempted to identify possible sites of action of two of the most potent protective flavonoids, epicatechin and kaempferol, in cultured primary neurons.

Endogenous galanin potentiates spinal nociceptive processing following inflammation.

We have undertaken a series of experiments using galanin null mutant mice to better define the role of endogenous galanin in spinal excitability following inflammation and in response to centrally sensitizing stimuli. We have employed a behavioural paradigm, the formalin test, as a model of tonic nociception in both galanin knock-out (gal-/-) and wild-type (gal+/+) mice. In this model, we find that gal-/- mice are markedly hypo-responsive, especially in the second phase response.

Antiparkinsonian activity and dyskinesia risk of ropinirole and L-DOPA combination therapy in drug naïve MPTP-lesioned common marmosets (Callithrix jacchus).

De novo administration of long-acting dopamine agonists, such as ropinirole, to patients with Parkinson's disease or to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated subhuman primates produces a lower incidence of dyskinesia than occurs with L-DOPA. This study compares the intensity of dyskinesia produced by combinations of L-DOPA and ropinirole and by these drugs alone, using the MPTP-treated common marmoset model of Parkinson's disease. The objective is to determine the optimum therapeutic strategy for the long-term control of Parkinson's disease with a minimal risk of dyskinesia.

Intrathecally delivered glial cell line-derived neurotrophic factor produces electrically evoked release of somatostatin in the dorsal horn of the spinal cord.

Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor with an established role in sensory neuron development. More recently it has also been shown to support adult sensory neuron survival and exert a neuroprotective effect on damaged sensory neurons. Some adult small-sized dorsal root ganglion (DRG) cells that are GDNF-sensitive sensory neurons express the inhibitory peptide somatostatin (SOM).

Effect of overexpression of wild-type and mutant Cu/Zn-superoxide dismutases on oxidative stress and cell death induced by hydrogen peroxide, 4-hydroxynonenal or serum deprivation: potentiation of injury by ALS-related mutant superoxide dismutases and protection by Bcl-2.

Mutations in Cu/Zn-superoxide dismutase (SOD1) are associated with some cases of familial amyotrophic lateral sclerosis (ALS). We overexpressed Bcl-2, wild-type SOD1 or mutant SOD1s (G37R and G85R) in NT-2 and SK-N-MC cells. Overexpression of Bcl-2 rendered cells more resistant to apoptosis induced by serum withdrawal, H2O2 or 4-hydroxy-2-trans-nonenal (HNE).

Selective activation of nociceptors by P2X receptor agonists in normal and inflamed rat skin.

1. ATP can elicit pain in humans and, together with other P2X channel agonists, can produce nocifensive responses in rodents. We used the rat in vitro skin-nerve preparation to quantify primary afferent responses to ATP and its stable analogue alpha,beta-methylene ATP in normal and carrageenan-inflamed skin.

Flavonoid antioxidants.

In order to ascertain the role of dietary flavonoids as antioxidants in vivo it is necessary to understand the chemical nature of the absorbed forms in the circulation in vivo and how the multiplicity of research findings in vitro reflect the bioactivity of flavonoids in vivo. Only when we gain adequate information on the circulating forms can we begin to understand the targeting to the tissues, whether flavonoids cross the blood-brain barrier, for example, and in what forms. Flavonoids are powerful antioxidants in vitro, but their overall function in vivo has yet to be clarified, whether antioxidant, anti-inflammatory, enzyme inhibitor, enzyme inducer, inhibitor of cell division, or some other role.

6-hydroxydopamine increases hydroxyl free radical production and DNA damage in rat striatum.

Oxidative damage is considered to be an important factor of 6-hydroxydopamine (6-OHDA) toxicity. To address this issue, microdialysis probes were implanted into the striatum of Wistar rats and perfused with 6-OHDA. Salicylate was included in the perfusion fluid to measure 2,3-dihydroxybenzoic acid (2,3-DHBA) as a marker of hydroxyl radical formation using HPLC with electrochemical detection.

Heterogeneity in nuclear transport does not affect the timing of DNA synthesis in quiescent mammalian nuclei induced to replicate in Xenopus egg extracts.

Intact G0 nuclei from quiescent mammalian cells initiate DNA synthesis asynchronously in Xenopus egg extracts, despite exposure to the same concentration of replication factors. This indicates that individual nuclei differ in their ability to respond to the inducers of DNA replication. Since the induction of DNA synthesis requires the accumulation of replication factors by active nuclear transport, any variation in the rate of transport among nuclei could contribute to the variability of DNA replication.

Epitope-tagged recombinant AAV vectors for expressing neurturin and its receptor in retinal cells.

Purpose: Neurturin (NTN) is a potent neuronal survival factor in the central and peripheral nervous systems. We previously described altered expression of mRNAs for NTN and one of its receptor components, GFRa-2 in degenerative retinas of rd/rd mice. Towards assessing the potential for transfer of these genes to counteract retinal degeneration, we examined recombinant adeno-associated virus (rAAV) constructs for expression of NTN and GFRa-2 transgenes in retinal cells in vitro and for the effect of transgene expression on retinal function following intraocular delivery in rd/rd mice.

Epicatechin and its in vivo metabolite, 3'-O-methyl epicatechin, protect human fibroblasts from oxidative-stress-induced cell death involving caspase-3 activation.

There is considerable current interest in the cytoprotective effects of natural antioxidants against oxidative stress. In particular, epicatechin, a major member of the flavanol family of polyphenols with powerful antioxidant properties in vitro, has been investigated to determine its ability to attenuate oxidative-stress-induced cell damage and to understand the mechanism of its protective action. We have induced oxidative stress in cultured human fibroblasts using hydrogen peroxide and examined the cellular responses in the form of mitochondrial function, cell-membrane damage, annexin-V binding and caspase-3 activation.

Bioavailability of flavonoids and potential bioactive forms in vivo.

Flavonoids are powerful antioxidants in vitro, but their overall functions in vivo have yet to be clarified, whether antioxidant, anti-inflammatory, enzyme inhibitor or inducer, or some other role. The reducing properties of flavonoids might also contribute to redox regulation in cells independently of their antioxidant properties. However, in order to understand their bioactivity in vivo, it is necessary to understand the factors influencing the absorption of flavonoids by the gastrointestinal tract, the nature of the conjugates and metabolites in the circulation and how this influences their antioxidant activities.